Combating cancer efficiently relies on pharmaceutical compounds directly targeting tumor cells or boosting host defense against said cells. Although several anti-cancer therapies are proposed, amongst which feature chemotherapy [anthracyclines, such as doxycycline (DOX), oxali-platinum (herein called PLAT) and cis-platinum (herein called PLAT) and tyrosine kinase inhibitors are considered as the most efficient cytotoxic agents of the oncologist armamentarium] and radiotherapy [X-Rays (XR)], the benefits of said treatments still tends to be insufficient. Since the above mentioned therapies represent the basis of up to 70% of anti-cancer therapies, detection of dysfunctions responsible for a reduced response to said treatments appears critical for patient management.
The emerging problem of therapeutic resistance of tumors underlines the importance to consider, firstly, the efficacy of the treatment on the tumor, and, secondly, the efficacy of the host immune system in the eradication of tumor cells. The inventors herein demonstrate that intrinsic (immune) tumor suppression mechanisms act in synergy with extrinsic tumor suppression mechanisms induced by cancer treatments, and that mutations in genes implicated in the immune response affect these extrinsic tumor suppression mechanisms. The mutational and transcriptional status of said genes constitute a new factor predictive of clinical response to cancer treatments.
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract1. GISTs are thought to originate from the neoplastic transformation of the interstitial cells of Cajal, the intestinal pacemaker cells. The true incidence of GISTs remains unknown, but experience from clinical trials suggests an incidence of 4500-6000 new cases per year in the United States2. The median age at diagnosis is approximately 58 years.
Historically, GISTs have been targeted by the three traditional cancer therapeutic modalities: surgery, chemotherapy, and radiotherapy. Surgery is effective for patients with resectable disease, but disease may recur in as many as 50% of individuals. Chemotherapy and radiotherapy have shown little efficacy3. A major breakthrough occurred in 1998 with the discovery of gain-of-function mutations in the KIT oncogene in GISTs4. KIT encodes the transmembrane KIT receptor tyrosine kinase (CD117) that, when activated via binding by its ligand, regulates the intracellular signal transduction process. Constitutive tyrosine kinase activation by mutation results in unregulated cell growth and malignant transformation. More than 90% of GISTs harbour activating KIT mutations5. These mutations commonly occur in exon 11 (juxtamembrane domain) in 57-71% of cases, exon 9 (extracellular domain) in 10-18% of cases, exon 13 (tyrosine kinase domain I) in 1-4% of cases, and exon 17 (tyrosine kinase domain II) in 1-4% of cases1. Approximately 35% of GISTs lacking KIT mutations have activating mutations in a gene encoding a related receptor tyrosine kinase, the platelet-derived growth factor receptor α (PDGFRA)6. PDGFRA mutations have been identified in exon 12 (1-2% of GISTs), exon 18 (2-6%), and exon 14 (<1%)7. Identification of KIT and PDGFRA mutations led to the development of specific targeted therapies with tyrosine kinase inhibitors (TKIs). Therapy with the TKIs imatinib mesylate (STI571, Gleevec-Novartis) and sunitinib malate (SU11248, Sutent-Pfizer) is effective for unresectable, metastatic, and recurrent disease8. Imatinib selectively inhibits several tyrosine kinases including KIT, PDGFRA, and ABL. Data from a phase II imatinib trial revealed that mutational status of KIT was the most important factor predictive of clinical response to imatinib8. Patients with GISTs expressing exon 11 KIT mutants who received imatinib had a substantially higher partial response rate, longer median survival, and less likelihood of progressing than those with GISTs expressing wild-type or exon 9 KIT mutants. Imatinib is a dramatically effective agent, but the duration of its benefits is finite. The second targeted tyrosine kinase inhibitor, sunitinib malate, has been approved for the treatment of imatinib-resistant GISTs after recent encouraging results9. However, as explained previously, drug resistance is an increasingly more common phenomenon10.
Responsible for 15% of all cancer-related deaths in children, neuroblastoma is the most common extracranial malignancy and second most common solid tumor affecting the paediatric population11. This cancer of the peripheral sympathetic nervous system is well known for its dichotomous pattern of presentation. Approximately one-half of children have localized tumors that can be cured with surgery alone. A small subset of children under 1 year of age who show disease involving the liver, skin, lymph nodes, or bone marrow have a good prognosis despite the extent of their disease. The remaining children have widespread metastatic disease or quite large, aggressive, localized tumors. These children have a poor long-term survival rate of approximately 30%. The challenge of treating children with neuroblastoma is to increase the survival of the high risk patients while avoiding overtreatment of those with lower risk disease. The standard prognostic indicators of outcome in neuroblastoma are age, stage, and histopathology. Additional chromosomal and molecular markers exist that are beginning to improve the accuracy of risk group assignment and outcome prediction12.
Among the actors of the anti-tumoral response, natural killer (NK) cells provide innate defence against tumors by virtue of potent capacities to immediately kill cellular targets and produce cytokines such as TNFα and IFNγ13.
NK cells control tumor growth by preventing the dissemination of metastatic tumors in mice14. The relevance of NK cells in human malignancies has been discussed by inventors in a recent Perspective written for Nature Immunol15. Recently published data obtained by inventors established in GIST patients that GIST tumors may be controlled by NK cells during the gold standard therapy with imatinib mesylate (IM). Indeed, IM can promote a c-kit dependent DC/NK cell cross-talk leading to NK cell IFNγ production in both mice and humans16. Importantly, the NK cell IFNγ production after 2 months of IM represented an independent predictor of long term survival in advanced GIST treated with IM17. Such an enhanced IFNγ production of purified NK cells was achieved after short term ex vivo restimulation with maturing DC, suggesting that NK cells from “immunological responders” had been primed in vivo during the GIST development18,19. Despite the ongoing questions regarding their effective role against human cancers, physiologic ligation of NK activating or cytokine receptors and/or blockade of inhibitory receptors can result in NK cell proliferation, trafficking, cytotoxicity, production of chemokines and cytokines and in dendritic cell triggering that can be exploited to harness hematological malignancies and some solid tumors.
The human major histocompatibility complex (MHC) encompasses about four megabases of DNA within the chromosomic region 6p21.3 that was characterized by a high density of polymorphic genes20,21. The telomeric end of the class III MHC has been designated as class IV region because it contains genes encoding inflammatory functions such as tumor necrosis factor (TNF) family members, allograft inflammatory factor 1 (AIF1), heat shock 70 kDa protein 1B (HSPA1B), lymphotoxin-α LTA) and -β (LTB), HLA-B associated transcript 3 (BAT3), leucocyte-specific transcript 1 (LST1), and natural cytotoxicity triggering receptor 3 (NCR3/1C7/CD337/NKp30)22 (cf. FIG. 1). The NCR3 gene is transcribed to several mRNA splice variants, most of which are translated into cell surface molecules of the immunoglobulin superfamily23-25. By generating monoclonal antibodies directed against the extracellular domain of the NCR3 gene product, Moretta's group succeeded in defining the cellular distribution of the NCR3 gene product and in characterizing NKp30 as a novel NK cell receptor involved in the killing of tumor cells and dendritic cells26,27. It is part of the so-called Natural Cytotoxicity Receptors (NCRs) also including NKp44, NKp46 and NKp8028. Beyond its expression in NK cells, NKp30 can be exposed on the surface of IL-15 stimulated umbilical cord T lymphocytes29 and endometrial epithelial cells30. However, in peripheral blood of the adult, the only circulating NKp30-expressing cells are NK cells. In contrast to NKp46, the murine ortholog of NKp30 is a pseudogene (in Mus musculus but not in Mus caroli)25,31,32 meaning that there is no suitable mouse model for the exploration of NKp30. However, rats express the gene NCR3 encoding an ortholog protein to NKp3033 and a transcript is detected in macaques (M. fascicularis)32.
Human NKp30 is a 190 amino-acids transmembrane protein with an extracellular variable-type Ig-like domain containing two putative N-glycosylation sites26. Three alternative splices can yield three different intracellular domains (that have 36, 25 and 12 amino acids) depending on which particular exon 4 they utilize24. An additional alternative splice can induce the deletion of 25 AA in the extracellular domain leading to the formation of a predicted constant-type Ig-like domain instead of a variable-type Ig-like domain24,32. In favour of the presence of multiple forms of NKp30 at the cell surface of NK cells, a western blot using a polyclonal antibody directed against NKp30 reveals a broad band of about 10 kD corresponding possibly to differences in glycosylation but also eventually to alternative protein cores26. Although these alternative forms of NKp30 were described, the functional relevance of this observation is not clear yet.
In regard to the role of NKp30 in tumor environment, NKp30 has been involved in the lysis of various tumours in vitro, including carcinomas, neuroblastomas, myeloid and lymphoblastic leukaemias and also in the lysis of various cell lines26,34,35. NKp30 is not only pivotal at the effector phase of immune responses (to attack targets) but also at the priming phase of cognate immunity. Indeed, NKp30 is regulating the cross-talk between DC and NK cells. Inventors were the first to describe the capacity of DC to trigger NK cell activation in mice36 and subsequent work definitely established the bidirectional regulation between these cell types27,37,38. Depending on the DC/NK ratio and on the KIR/NKG2A expression on the NK cell side, activated NK cells may either kill or promote the immature DC activation. In both cases, NKp30 is directly involved in the recognition of DC by NK cells. In the latter case (when activated NK cells activate immature DC), NKp30 triggering leads to TNFα and IFNγ release by NK cells, both cytokines participating in the DC maturation process39. Hence, the DC/NK cell cross talk appears to be critical to modulate Th1 differentiation40,41. Therefore, NKp30 triggering could represent a master regulator of the adaptive immune responses in the lymph nodes.
Recent studies have demonstrated a role of NKp30 in human malaria infection42,43. The results suggest that NKp30 is involved in the NK cell-Plasmodium falciparum-parasitized red blood cells interaction42. This interaction is direct, specific and functional, leading to perforine production and granzyme B release. Moreover, an association has been demonstrated between mild malaria and a mutation located within the NCR3 promoter (NCR3-412 G/C-rs2736191)43. This suggests that genetic variation in NKp30 may account for the heterogeneity of human NK cell reactivity to P. falciparum-infected erythrocytes44.
It has been suggested that NKp30 could recognize heparan sulfate residues on the cellular membrane of target cells, either tumor cells or dendritic cells45. However, these results were criticized by Warren et al46. The group of Angel Porgador tried to settle this controversy by comparing various NKp30Fc recombinant proteins used in these studies. Their findings are that glycosylations on the Fc proteins are important in the recognition of NKp30 ligands and they confirm their original findings that heparan sulfates bind to recombinant NKp30 in in vitro assays47. Pogge et al claims that Leukocyte Antigen-B-associated transcript 3 (BAT3) is released by tumor cells and engages NKp3048. Byrd et al demonstrated that NKp30 ligands are expressed intracellularly in most cell lines and probably in early endosomal compartments49.
In summary, strong evidence lack on the exact nature of NKp30 ligand(s), in particular in tumoral environment and in in vivo models.